Effect of magnesium supplementation in improving hyperandrogenism, hirsutism, and sleep quality in women with polycystic ovary syndrome: A randomized, placebo‐controlled clinical trial

Abstract Background and Aims Polycystic ovary syndrome (PCOS) is one of the common endocrine disorders in women, which causes numerous symptoms in women. The relationship of many micronutrients with this syndrome has been investigated. This study was conducted to examine the effects of magnesium supplementation on hyperandrogenism, hirsutism, and sleep quality in women with PCOS. Methods In this parallel randomized clinical trial, 64 women with PCOS were randomly assigned to the magnesium group (n = 32) or placebo group (n = 32) for 10 weeks. Patients in the magnesium group received one 250 mg magnesium oxide tablet, per day. Hyperandrogenism, hirsutism, and sleep quality were measured at the beginning and end of the study. This randomized clinical trial was registered at https://www.IRCT.ir (IRCT20130903014551N8). Results Magnesium supplementation had no significant effect on hyperandrogenism (p = 0.51 for dehydroepiandrosterone sulfates, p = 0.27 for testosterone), hirsutism (p = 0.23), and sleep quality (p = 0.85) compared with placebo. Conclusions The present study showed that a single dose of magnesium supplementation elicited no beneficial effects on the mentioned symptoms in polycystic women. It is possible that the positive effects of magnesium observed in the former studies were due to the synergistic effects of other vitamins or minerals. More studies are needed in this area.


| INTRODUCTION
Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women. The prevalence of PCOS is about 4%-21%, depending on the different diagnostic criteria. 1 Women with PCOS have several symptoms including hirsutism, hyperandrogenism, and sleep disorders that affect the quality of life. 2 However, it seems that some nutrients may have a role in the improvement of these conditions. Magnesium is a fundamental mineral in the human body, considered a common intracellular electrolyte, 3 and is necessary for the function of 300 enzyme systems involved in energy metabolism and nucleic acid synthesis. 4 Recent evidence has suggested that women with PCOS may have lower levels of serum magnesium compared with healthy women 5,6 ; in addition, it has been reported that magnesium may contribute to a reduction of PCOS symptoms. Some studies have shown that, in women with PCOS, serum magnesium concentration was higher in individuals without hirsutism compared with women with hirsutism. 7 Also, an improvement in hirsutism was observed after cosupplementation of magnesium and vitamin E in women with PCOS. 8 Serum magnesium levels may also be associated with hyperandrogenism 7 ; indeed, a cohort study reported that insufficient dietary intake of magnesium was related to hyperandrogenism among women with PCOS. 9 Similarly, there was an inverse association between serum magnesium and testosterone concentration among patients with PCOS. 10 On the other hand, magnesium supplementation significantly reduced sleep disorders in the elderly, 11 and a systematic review and meta-analysis showed that oral magnesium supplementation may improve insomnia and sleep quality in older adults. 12 There is a lack of enough evidence regarding magnesium supplementation in women with PCOS. Indeed, previous studies have not focused on the effect of magnesium supplementation on sleep quality and hyperandrogenism in these patients, and although some studies examined the impact of magnesium supplementation on hirsutism, such studies used magnesium in combination with other nutrients and did not evaluate the effect of magnesium alone on hirsutism in PCOS. Therefore, the aim of the present clinical trial was to evaluate the effect of magnesium supplementation on hirsutism, hyperandrogenism, and sleep disorders in women with PCOS.

| Participants and randomization
The present study was a parallel double-blind randomized controlled trial, with a duration of 10

| Intervention
Patients received sufficient information regarding the study. Women in the magnesium group received a 250 mg magnesium oxide tablet (Magni One ® produced by DonyaDarou) per day for 10 weeks.
women in the placebo group received a tablet containing 5 mg starch, and its color, appearance, smell, and taste were similar to the magnesium tablet. Participants in both groups were recommended to take the tablets after breakfast. Participants were monitored through a telephone call and virtual networks, and they were asked to give back empty containers of supplements to ensure that all supplements were used properly.
A list of following dietary recommendations was provided for participants in both groups: (1) to avoid consuming refined or simple carbohydrates, such as white bread, white rice, sugar, and sweets; (2) consume higher amounts of fresh vegetables; (3) increase the number of meals and reduce the volume of each meal; (4) drink adequate fluid, especially water (at least eight glasses per day); (5) replace starchy vegetables, such as potatoes with nonstarchy vegetables, such as lettuce, cabbage, cucumber, and so on; one study has shown that consuming a low starch diet such as low starch carbohydrates resulted in weight loss, improved insulin sensitivity, and reduced testosterone in women with PCOS 15 ; (6) consume fresh fruits instead of industrial and sugar-sweetened fruit juices; (7) avoid consuming salty foods, fast foods, and high-fat dairy products; and (8) consume healthy oils, such as olive oil and canola oil, instead of saturated or partially saturated vegetable oil, or animal fat.

| Biochemical assessment
The serum concentration of magnesium, DHEAs, and testosterone was measured at baseline, and DHEAs and testosterone were remeasured after 10 weeks. A 10 ml blood sample was drawn, and after serum isolation, magnesium was measured by the atomic absorption spectrophotometry method. Also, radioimmunoassay and high-performance liquid chromatography were used to measure the level of testosterone and DHEAs, respectively. Each domain was scored from 0 to 3 points, and the total score of the PSQI sleep quality questionnaire ranged from 0 to 21, where higher scores indicated lower sleep quality. A score greater than 5 indicates that the person was sleeping less. 16

| Assessment of hirsutism
A modified Ferriman-Gallwey questionnaire was used to assess the hirsutism status and was completed by a trained staff. The validity and reliability of this questionnaire were previously evaluated and shown to be acceptable. 18 In this questionnaire, the density of hairs at three sites (upper lip, thigh, and lower abdomen) were measured. A score of 0-4 was given based on the density of the terminal hairs. A score higher than 4 indicates the presence of hirsutism.

| Assessment of dietary intake and physical activity
To control the confounding effect of dietary intake and physical activity, five 1-day food diaries and five 1-day physical activity records were completed by each participant during the study.
Records covered three usual days and two weekend days. Patients were visited at baseline, the 5th week, and at the end of the study.
Food diaries and physical activity records were provided during these visits. Food records were analyzed using Nutritionist IV designed based on the USDA food composition database. Individuals were asked to report their activities, including walking, exercise, sleep, hours spent watching TV, housework, studying, bathing, and other activities, and subsequently, the total metabolic equivalent (MET) was calculated by multiplying the frequency, duration, and intensity of each physical activity in 24 h. 19 Physical activity was reported in terms of MET per hour (MET/h/day).

| Statistical analysis
An intention-to-treat (ITT) analysis based on the linear regression method was applied in the present study. 20 Normal distribution was evaluated by using the Kolmogorov-Smirnov test and visual inspection of Q-Q plots, and we found that the distribution of DHEAs was not normal. Therefore, a log transformation method was used to attenuate non-normal distribution. We compared qualitative variables between the magnesium and placebo groups using the 3 | RESULTS Therefore, 54 patients completed the study. By using an ITT method, data from 64 participants (n = 32 in each group) were included in statistical analysis.
The general characteristics of the participants are shown in Table 1. Accordingly, results revealed that age (p = 0.62), weight However, baseline serum magnesium levels were significantly higher in the magnesium group than in the placebo group (p = 0.04). Table 2 shows the energy-adjusted dietary intake of participants during the study. There were no differences in dietary intake between the two groups.
The effects of magnesium supplementation on hyperandrogenism, hirsutism, and sleep quality are shown in Table 3. DHEAs   The results of the present study revealed that magnesium supplementation could not change sleep quality. Although a clinical trial reported an improvement in insomnia following magnesium supplementation, magnesium was used in combination with melatonin and vitamin B complex. 24 Moreover, the results of an observational study showed no associations between dietary magnesium intake and daytime sleepiness nor night snoring in men and women. 25 A systematic review summarized the effect of oral magnesium supplementation on insomnia in older adults and reported that there was insufficient evidence to advocate the prescription of magnesium as a complementary therapy for insomnia. 12 As mentioned, the present study did not observe a significant effect of magnesium supplementation on the improvement of hyperandrogenism, hirsutism, and sleep quality in women with PCOS. Some possible reasons for these nonsignificant findings are: (1) Most studies that reported a significant effect of magnesium supplementation used cosupplementation of magnesium and other nutrients. Therefore, it seems that synergistic interaction between nutrients may have contributed to beneficial effects. For example, vitamin E and magnesium are known to have a synergistic interaction and strengthen each other in biological pathways. 26,27 Also, vitamin B6 has synergistic effects on magnesium function, 28 and the synergistic interaction between vitamin C and vitamin B12 and magnesium, respectively has been approved. 29 analysis, and interpretation of data; writing of the report; and the decision to submit the report for publication.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
Data are available on request from the authors. Obtained from an independent t-test comparing endpoint measurements between two groups. d Obtained from analysis of covariance, adjusted for baseline value of each factor and baseline serum magnesium comparing endpoint values between two groups.